Abstract APOBEC family cytidine deaminases are prominent mutators of cancer genomes, often causing thousands of C to T base substitutions in affected tumors. These enzymes normally function within the immune system and lipid metabolism. How APOBECs become dysregulated and the role subsequent of APOBEC-induced genetic instability has in promoting cancer progression are unclear. Also unknown is whether APOBECs induce other types of genetic instability in tumors in addition to base substitutions caused by deamination. The overall objective of this proposal is to identify mechanisms that enable APOBEC mutagenesis, determine how this activity alters cancer genomes, and assess the contribution of this genetic instability to carcinogenesis. Aim 1 will determine if increased levels of single strand DNA caused by chemical and oncogene-induced replication stress facilitates APOBEC mutagenesis. We will measure the effects of replication stress on APOBEC-induced mutation frequencies in human cells, determine if cancer cells displaying endogenous APOBEC activity also display markers of replication stress, and determine whether replication stress induced by Her2 activation synergizes with APOBEC mutagenesis to alter cell proliferation and accelerate tumorigenesis in a mouse model. Aim 2 will address how deficiencies in chromatin modifiers influence APOBEC mutagenesis. We have determined that loss of chromatin modifying enzymes, which are also frequently inactivated during cancer development, increase the frequency of APOBEC-induced mutations in yeast. The mechanisms underlying this effect will be determined and re-capitulated in human cells. Additionally, associations between chromatin modifier loss and elevated numbers of APOBEC-induced mutations in sequenced cancers will be bioinformatically evaluated. Aim 3 will investigate whether APOBECs are capable of inducing chromosomal rearrangements during cancer development. We will measure APOBEC-induced frequencies of non-allelic recombination and copy number variation in yeast and human cells as well as evaluate whether APOBECs mutagenize the single strand DNA intermediates formed during these recombination events to cause kataegis. The results from the experiments proposed in these Aims will likely demonstrate that mutation-driven, cancer-associated changes in DNA metabolism increase APOBEC- induced mutagenesis, investigate an expanded role for APOBECs in promotion of additional types of genetic alterations, and directly characterize the ability of APOBEC-generated mutagenesis to promote tumorigenesis. Successful completion of these aims will describe a critical aspect in the development and progression of a significant number of human tumors.